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Hfr FORMATION DIRECTED BY TnlO FORREST G. CHUMLEY, ROLF MENZEL AND JOHN R. ROTH

Department of Biology, University of Utah, Salt Lake City, Utah 84112 Manuscript received July 18, 1978 ABSTRACT

The transposable drug-resistance element, TnlO, can serve as a region of homology to direct the insertion of an F’tsll4 lac plasmid into the chromosome of Salmonella typhimurium. Derivatives of F’tsl14 lac were constructed that carry TnlO insertions; these plasmids were transferred to strains having a TnlO insertion in the chromosome. Under these circumstances, Hfr formation requires homologous recombination between plasmid-borne and Chromosomal TnlO elements. The process is dependent on recA function and on the presence of both TnlO elements. All Hfr’s isolated from a given merodiploid show the same direction of transfer. Depending on the orientation of TnlO in the F’ plasmid, Hfr’s transferring in either direction can be obtained from any chromosomal TnlO insertion. Since TnlO insertions can be generated in any region of the chromosome, this method permits the isolation of Hfr’s with either direction of transfer having their origin at almost any predetermined site. The Hfr’s constructed by this method are sufficiently stable for standard genetic mapping crosses, and they have also been used to generate new F’ plasmids. Implicit in the results above is the possibility of determining the orientation of any chromosomal TnlO insertion by constructing an Hfr using a standard F TnlO plasmid and determining the direction of chromosome transfer. The general approaches described here are applicable to other transposable elements and other bacterial systems.

T H E . l a c k of an appropriate Hfr has occasionally limited the utility of bacterial conjugation in genetic mapping and has made it difficult to derive F’ plasmids carrying certain genes. We have developed a method using the transposable drug resistance element, TnlO (KLECKNER et al. 1975; KLECKNER, ROTHand BOTSTEIN1977), which permits the isolation of an Hfr with an origin at essentially any selected site on the chromosome. The method also permits selection of the direction of chromosome transfer. This method should be valuable for E . coli and Salmonella, but it should be particularly useful in genetic systems for which few Hfr’s have been characterized. We have used newly formed Hfr’s with origins near the genes for proline utilization ( p u t ) in the derivation of F’ plasmids that carry the put region. The method for Hfr formation involves the directed insertion of an F’tsll4 lac plasmid into the chromosome by recombination between a TnlO sequence carried on the plasmid and a second TnlO sequence located on the chromosome, as diagrammed in Figure I.This is an extension of the method described by CUZIN and JACOB (1964) and by BECKWITH, SIGNERand EPSTEIN (1966) for the transposition of the lactose operon in E . coli. Wild-type S. typhimurium lacks the Genetics 91 : 639-655 April, 1979.

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F. G. CHUMLEY, R. MENZEL AND J. R. ROTH

i l x lacf

F l s lac z z f - 2 0 : : T n K

w A

FIGURE 1.--A diagrammatic representation of Hfr formation. Recombination between TnlO sequences carried by both the plasmid and the chromosome integrates F‘tsll4 lac (JACOB, BRENNERand CUZIN 1963; CUZIN and JACOB 1967) into the chromosome. The TnlO elements are represented by wavy lines, and a n arrow indicates the orientation of each insertion. For the Hfr formed, the origin of transfer is between the markers a and b; marker b will be transferred early. If the orientation of either TnlO insertion were reversed, the resulting Hfr would transfer a early, instead of b.

genes for the utilization of lactose, but it is Lac+ when it harbors a plasmid carrying the lac operon of E. coli, such as F’tsll4 lac. This particular plasmid BRENNERand CUZIN 1963; is temperature sensitive for replication (JACOB, CUZINand JACOB 1967), and it is readily lost during growth at 40°, conferring a temperature-sensitive Lac+ phenotype on the Salmonella host cell. When Lac+ survivors are selected at 40°, the most common class results from integration of the F’ts114 lac plasmid into the chromosome and its passive replication along with the chromosomal DNA (BECKWITH, SIGNERand EPSTEIN1966). The point of F’ insertion determines the origin of an Hfr; the orientation of the homologous chromosomal and episomal regions during the insertion event determines the direction of chromosome transfer. The TnlO element is a 9,200 base-pair DNA sequence that determines resistance to the antibiotic, tetracycline. The entire element can transpose as a unit in a process that does not depend on homologous recombination (KLECKNER et al. 1975). TnlO insertion mutations within or near any genes of interest can be recovered by rather simple procedures (KLECKNER, ROTH and BOTSTEIN 1977). We have isolated and characterized a number of these insertions in the his and put regions, in addition to a number of insertions into the F’128 pro lac plasmid. Among the insertions into F’128, some are located in F-specific sequences, and some are located in bacterial chromosomal sequences. TnlO can insert in either of two orientations, and our method for Hfr isolation permits us to determine the relative orientation of any individual insertion, simply by observing the direction of chromosome transfer of Hfr’s formed by recombina-

H F R FORMATION DIRECTED BY T N I O

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tion between a given chromosomal TnlO element and a TnlO element carried by the F' plasmid. The orientation of the chromosomal TnlO insertion can be confirmed by using a series of F' plasmids carrying TnlO in different orientations. All the Hfr's we have isolated from any given merodiploid transfer the chromosome in the same preferred direction. Although this work has been done in Salmonella, the procedures should be applicable to other transposable elements and other bacterial systems, where they might facilitate otherwise difficult genetic analyses. MATERIALS .4ND M E T H O D S

Bacterial strains: The strains used for this study are derivatives of Salmonella typhimurium LT2. Table 1 lists the numerical designations and full genotypes of multiply marked strains. Unless otherwise indicated, the isolaticn or construction of strains is described in this paper. Strains bearing the prefix "TT" either contain the transposable tetracycline resistance element, TnlO, or they were derived from parental strains that contained TnlO. Media: Difco nutrient broth (8 g/l), with 5 g per 1 NaCl added, was used as rich medium. The E medium of VOGELand BONNER(1956), containing 2% glucose, was routinely used as minimal medium. To select for growth on lactose as the sole carbon source, the NCE (modified E) medium described by BERKOWITZ et al. (1968) was used, supplemented with 0.2% lactose, final concentration. To select for growth on 0.2% proline as the sole nitrogen source, strains were grown on the NCN-succinate medium described by RATZKIN,GRABNAR and ROTH (1978). For use in plates, medium was solidified by the addition of Difco agar to 1.5%. When required, tetracycline (Sigma) was added to a final concentration of 25 gg per ml in rich medium o r 10 pg per ml in minimal medium. Lactose tetrazolium indicator plates (LEDERBERG 1948) contained, per liter, 23 g Difco nutrient agar, 1 g NaCI, 50 mg Difco Bacto-TTC, and, finally, 50 ml of a filter-sterilized 20% lactose solution added after autoclaving the other ingredients. Transduciional methods: Bacteriophage P22 containing the mutations HT105/1 (SCHMIEGER 1971), which causes an increased frequency of generalized transduction, and int-201 (ANDERSON and ROTH1978), which prevents stable lysogen formation, was used for all transductions. Phage were grown on donor strains as described by SCOTT,ROTHand ARTZ(1975). In most crosses, phage and bacteria were mixed directly on selective media. When selection was made for inheritance of a donor TnlO, phage and bacteria were mixed and preincubated for 30 min in a nonselective 1iqu;d medium before plating on tetracycline-containing media. Transductant clones were purified and made phage-free by streaking alternately on rich and selective media. Construction of merodiploids conidning chromosomal and plasmid-borne TnlO insertions: F'tsll4 lac plasmids carrying various TnlO insertions were transferred from p y r strains TT627, TT628, and TT629 into recipients containing TnlO insertions in the put or his regions and a counter-selective auxotrophic marker. The donor strains were grown selectively a t 30" in NCE medium containing lactose and uracil, and Lac+ transconjugants were selected a t 30" by crossstreaking donor and recipient rJn lactose plates supplemented as required by the recipients, but containing no uracil. These merodiploids were purified by streaking on the same selective medium. In all these merodiploids, the Lac+ character was stable at 30°, but was lost a t high frequency when ihe